The overall objective of this proposal is to gain further understanding of the role of the endothelial cell in disease by elucidating mechanisms involved in initiation and regulation of apoptosis. Endothelial cell apoptosis can occur in response to numerous environmental stimuli and has been implicated in several pathologic conditions. Infection of endothelial cells with the obligate intracellular bacterium, Rickettsia rickettsii, will be used as a model system. The endothelial cell is the primary target during human infection, which results in the disease known as Rocky Mountain spotted fever, and endothelial cell responses to intracellular R.rickettsii likely play an important role in the pathological events that characterize this disease. Intracellular infection with R.rickettsii likely play an important roles studies to transduce both a pro-and anti-apoptotic signal. Blocking activation of the transcription factor, nuclear factor-kappaB, during infection results in rapid host cell apoptosis. This provides a valuable pathophysiologically-relevant model system for study of the mechanisms involved in regulation of this cellular response. The studies described in Specific Aim 1 are designed to further characterize the R. rickettsii-induced apoptotic program, including study of kinetics, dependence on intracellular infection and critical aspects of the infection required for its initiation. Specific Aim 2 will explore involvement of known signal transduction pathways or events including generation of reactive oxygen species, caspase activation and p53. Specific Aim 3 will explore will explore the consequences of endothelial cell activation by R. rickettsii in the context of the naturally- occurring disease setting, wherein it will be determined if infection protects the endothelial cell from exogenous pro-apoptotic stimuli which may be generated by inflammation or immune cell recruitment Microscopic techniques will also be used to explore the correlation between intracellular infection, apoptosis infection and activation of nuclear factor-kappaB both in cell culture models and in an ex vivo model of infection. These studies will not only provide important insight into regulation of endothelial cell apoptosis, but also into the complex interplay of signaling events which occur as part of the host cell- parasite relationship.